The broad, long term objective of the proposed studies is to design and engineer phosphonic acid biosynthesis with the ultimate aim of developing a new framework for economical production of a wide variety of natural and unnatural natural products in Escherichia coli. Secondary metabolites - natural products - produced by plants and microorganisms are a prolific source for pharmaceutical drugs. Recent advances in molecular biology and genomics have revolutionized our ability to discover the biosynthetic pathways that synthesize natural products. However, since plants and microorganisms that produce interesting natural products are often poorly characterized or even uncultivable, the ability to manipulate their biosynthetic pathways is rather limited. In addition, natural products have evolved for the purpose other than treatment of human diseases. Although many natural products exhibit a diverse array of potent biological activities, their therapeutic efficacy often needs to be improved by further diversifying the core structures. More importantly, many of the natural product based drugs are produced in minute amounts in their native hosts, making the drugs very expensive. To address these issues, this proposal will use two important phosphonic acid (fosfomycin and FR-900098) biosynthetic pathways as model systemsto develop new methods and tools for economical biosynthesis of phosphonic acid antibiotics in E. coli. Such tools and methods should be generally applicable to the economical biosynthesis of a wide variety of natural products for biomedical applications. Moreover, microbial overproduction of FR900098, a promising antimalarial drug, may provide sufficient amounts at a low price for use in malaria-afflicted regions in the world. Finally, as a synergistic component of the program project, all relevant enzymes from these two pathways as well as other phosphonic acid (phosphinothricin, A53868, plumbemycin, rhizocticin, and SF2312) pathways will be overexpressed and purified for X-ray structural analysis by the Nair group. Some of them will also be biochemically and mechanistically characterized by the Metcalf, van der Donk and Kelleher groups.